Three New Species and a New Record of the Lichen Genus Peltula (Peltulaceae) from Helan Mountain in China

Simple Summary This paper reports on three new species and a new record of the lichen genus Peltula Nyl., highlighting research progress on lichen diversity in Helan Mountain. In this study, species were identified through a combination of morpho-anatomy, molecular systematics, and chemical substance detection. The results of the study enrich the data of Peltula and the study of lichens in Helan Mountain. Abstract In this study, a systematic taxonomic analysis was carried out on the lichen genus Peltula, collected from Helan Mountain in China; three new species (Peltula helanense, P. overlappine, and P. reticulata) and a new record (P. crispatula (Nyl.) Egea) for China were identified. Four species were identified by morph-anatomical, chemical, and phylogenetic analyses by combining two gene loci (ITS and LSU). Peltula helanense is with tiny individual thalli up to 1mm, attached by creamy-white cylindrical rhizoids and apothecia filling the whole squamule. Peltula overlappine is characterized by thallus squamulose forming rosette-shaped patches and squamules with distinctive thickened margins. Peltula reticulata is characterized by brownish brown thallus and squamules with densely reticulate upper surface. P. crispatula is characterized by irregular squamules attached to a tuft of hyphae. The four species are described in detail, compared, and discussed with similar species, and images of morpho-anatomical structures of the four species are also provided. Moreover, a key to the species of Peltula from Helan Mountain is provided. The results enrich the data of the genus Peltula and also indicate that the rich diversity of lichen species in Helan Mountain is worthy of in-depth study.


Introduction
The lichen genus Peltula (Peltulaceae, Lichinales, Lichinomycetes, Ascomycota) was established by Nylander [1], with Peltula radicata Nyl. as the type species.After the establishment of Peltula, the genus was not further used for a long time, and many species now belonging to Peltula Nyl. had been included in Heppia Naeg.Until Gyelnik's [2] revision of Heppiaceae, Peltula was used as a separate genus again.Later, Peltulaceae was established by Büdel [3], based on the type of ascocarps development, ascus characteristics, etc., and Peltula was moved into this family.According to the recent revision of the Peltulaceae by Frank et al. [4], Peltulaceae is verified as a monophyletic family through phylogenetic analyses, and Phyllopeltula and Neoheppia were moved into Peltula.At this point, the family Peltulaceae consists of only one genus Peltula.A total of 68 species of Peltula have been reported worldwide (https://indexfungorum.org/, accessed on 8 June 2024).
Helan Mountain is a concentrated distribution area of endemic plants in the arid de-sert area in the middle of the Asian continent.With rich plant species and complex flora, it is a rare treasure trove of biological resources and an important center of biodiversity evolution in the arid region of northwestern China [18].Peltula is a part of the lichen resources of Helan Mountain, mainly distributed in the premontane desert grassland area.It plays a vital role in stabilizing the land surface in the desert grassland area.In this study, we conducted a systematic taxonomic study of the genus Peltula collected from Helan Mountain.Eleven species were identified, including three new species (Peltula helanense S.Y.Wang and D.L. Niu, P. overlappine S.Y.Wang and D.L. Niu, and P. reticulata S.Y.Wang and D.L. Niu) and a new record (P. crispatula (Nyl.)Egea) from China.

Taxon Sampling
All specimens in this study were collected from Helan Mountain of China and preserved in NXAC (Botanical Herbarium, College of Life Sciences, Ningxia University, Yinchuan, China).The external morphology of the thallus and reproductive structures was observed, measured, and photographed using a stereo microscope (Soptop SZX12, Sunny Optical Technoligy (group) Co., Ltd., Ningbo, China); the anatomical structures were observed, measured, photographed, and recorded under electron microscope (Soptop RX50, Sunny Optical Technoligy (group) Co., Ltd., Ningbo, China).Microstructural preparations of thallus and reproductive structures were obtained using a freezing microtome (LEICA CM1950, Leica Holding B.V., Wetzlar, Germany).Some samples were sliced freehand by wetting the sample with distilled water and then cutting the thallus with a razor blade.A chemical color reaction was performed using 10% KOH aqueous solution, Ca(ClO) 2 saturated aqueous solution, and 10% Lugol's iodine solution.Lichen secondary metabolites were analyzed using TLC in system C (toluene: acetic acid = 170:30) [19].

DNA Extraction, Amplification, and Sequencing
Seven fresh specimens were selected for DNA extraction.ITS and LSU sequences were amplified by PCR.Genomic DNA was extracted using the Biospin Plant Genomic DNA Extraction Kit (Bruker Biospin Corporation, Etlingen, Germany).ITS was amplified using the primers ITS1F [20] and ITS4 [21,22]; LSU was amplified using the primers LR0R and LR7 [4].The PCR mixture (25 µL) contained 25 µg of BSA, 1 U Taq DNA polymerase, dNTP (0.2 mmol/L), primers (0.5 µmol/L each), and PCR buffer, which was replenished to 25 µL with H 2 O. PCR cycling parameters consisted of the initial denaturation at 94 • C for 2 min, followed by denaturation at 94 • C for 30 s, annealing at 55 • C for 30 s, extension at 72 • C for 70 s, and finally, 72 • C extension for 2 min, and the amplification products were stored at 4 • C. The products were purified, identified, and sequenced using an ABI3730XL automated sequencer.The new sequences generated in this study were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/,accessed on 12 March 2024).

Sequence Alignment and Phylogenetic Analysis
Based on bidirectional sequencing results, sequence splicing was performed using Sequencher 4.1.4with manual correction.In total, 120 reference sequences were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genbank/,accessed on 12 March 2024) and phylogenetically analyzed with the 15 sequences in this study (Table 1), and Peccania sp. and Peccania terricola were selected as outgroups.In PhyloSuite [23], sequence alignment was performed using MAFFT V 7 for ITS and LSU sequences separately [24], and Gblocks were used to eliminate uncertain regions [25].Sequences were concatenated using the software's concatenate function.The nucleotide substitution model before tree building was selected using ModelFinder [26].The GTR+F+I+G4 model was chosen in Bayesian analyses and repeated for 700,000 generations, sampling every 1000 steps to construct the MrBayes phylogenetic tree [27].The tree was aligned and beautified in iTOL (https://itol.embl.de/,accessed on 25 March 2024).

Phylogenetic Analysis
A total of 135 DNA sequences were used, including 15 new sequences (6 ITS, 9 LSU) (Table 1).In total, a data matrix corresponding to the ITS and LSU regions was generated with 1650 characters (ITS, 1-359; LSU, 360-1650).Bayesian Inference (BI) phylogenetic trees of the concatenated data set of the two gene markers were constructed (Figure 1).part of the taxon, it is morphologically fully characterized as a species of Peltula.It has small squamules with ascus with lacerated gelatinous sheath, and ascospores are singlecelled, colorless, and transparent.

TLC Results
Chemical substances of four species were detected by TLC.The results show four species containing cyathomorpha-unknown, an unknown fatty acid (Rf = 48), and a terpenoid (Rf = 46), which distinguished previous studies (Figure 2).P. overlappine also contains a yellow-green pigment (Rf = 25.5).The resulting tree showed two well-supported branches (PP = 1, BS = 100) corresponding to the new species Peltula helanense and the new record species P. crispatula.Moreover, almost all species had strong support.P. reticulata and P. polyspora are well-supported (PP = 1), but it is easy to distinguish them by their appearances.P. overlappine and P. patellata are also well-supported (BS = 91).However, the morphology and chemistry of the two species were distinct.Although the new species P. helanense is at the outermost part of the taxon, it is morphologically fully characterized as a species of Peltula.It has small squamules with ascus with lacerated gelatinous sheath, and ascospores are single-celled, colorless, and transparent.

TLC Results
Chemical substances of four species were detected by TLC.The results show four species containing cyathomorpha-unknown, an unknown fatty acid (Rf = 48), and a terpenoid (Rf = 46), which distinguished previous studies (Figure 2).P. overlappine also contains a yellow-green pigment (Rf = 25.5).Diagnosis: Tiny, up to only 1 mm, creamy-white cylindrical rhizoids, with apothecia of the mature occupying almost the entire upper surface of thallus.The side of thallus with umbilicus looks like an open umbrella.
Ecology and distribution: P. helanense is harvested from Helan Mountain in China.It grows on rocks and occurs only in China.
Notes: P. helanense is morphologically very similar to P. obscurans var.deserticola in that both thallus are small, squamulose, rounded or suborbicular, and have red and immersed apothecia.But P. helanense has only about 64 ascospores, whereas P. obscurans var.deserticola has more than 100.P. obscurans var.deserticola adheres to the substrate mainly by rhizines and umbilicus [5,6], while P. helanense is attached to the substrate by creamy-white cylindrical rhizoids.
Additional specimens examined: China, Ningxia Prov, Helan Mountain, Maliankou, 1682.5 m elev., on rock, 38°32′8.8″N, 105°54′27″ E, 21 September 2014, D.L. Niu, S.L. Ma, F. Chen, and G.M. Zhang.DNA voucher: 14020467.Notes: P. reticulata is closely related to P. polyspora in the phylogenetic tree, but the two species are morphologically distinct.P. reticulata is brownish, reticulately lobed from juvenile to mature, bluntly serrated on the underside of the margins, no pruinose, and with a 20-30 µm hyaline lamina in the outermost layer.In contrast, P. polyspora is olive-greenish brown, with smooth, entire margins, rarely lobed, yellowish pruinose, and without a hyaline layer.And P. reticulata has more spores than P. polyspora [11].In contrast to the dense, deep fissures of mature P. impressula, which are more profound, the more mature and more significant the individual, juveniles have no fissures.P. reticulata is uniformly fissured and cracked from juvenile to mature.

Discussion
Four species of Peltula growing in Helan Mountain were described.The genetic relationships between species of Peltula were revealed in phylogenetic analyses using two loci as markers.DNA sequences can explain phylogenetic relationships for species that are difficult to define by relying on morphology.P. overlappine, P. patellata, P. reticulata, and P. polyspora all show high posterior probabilities in the phylogenetic tree but are morphologically quite different.This study concludes that morphological observations must be combined with molecular data to identify lichen species more accurately.
Helan Mountain has been seriously neglected in lichen research.In addition to the four species recorded in the text, we found seven other reported species of Peltula.This suggests that Helan Mountain lichens are rich in species and a hotspot for lichen distribution.Among them, three species (P.africana, P. impressa, P. helanense) were found on rocks, while the remaining eight species (P.crispatula, P. euploca, P. impressula, P. overlappine, P. radicata, P. reticulata, P. richardsii, P. subpatellata) grew on the soil surface and formed biological soil crusts.It is assumed that this is related to the geographic environment of Helan Mountain, where most of the species in this study were collected from the premontane desert steppe area, where the arid climate with little rainfall, sparse vegetation, and calcium-rich soil provides a favorable environment for the growth of lichens of Peltula.

Conclusions
In this study, three new species (Peltula helanense S.Y.Wang and D.L. Niu, P. overlappine S.Y.Wang and D.L. Niu, and P. reticulata S.Y.Wang and D.L. Niu) and a new record species (P.crispatula (Nyl.) from China are described from Helan Mountain in northwest China.At present, we have identified a total of 11 species (P.africana, P. crispatula, P. euploca, P. impressa, P. impressula, P. helanense, P. overlappine, P. radicata, P. reticulata, P. richardsii, P. subpatellata) of Peltula distributed in Helan Mountain.This suggests that Helan Mountain is by far the richest area in China in terms of lichen species diversity in Peltula.Based on this study, the importance of lichen research in Helan Mountain is also demonstrated.

Future Prospectives
We are continuing to delve deeper, with some specimens yet to be identified.We will continue to study the species composition and ecological distribution of Peltula in the desert steppe area and which species dominate biological soil crusts.

Figure 1 .
Figure 1.Phylogram based on ITS and LSU sequences of Peltula species; the MrBayes tree is shown with posterior probability (PP) values and bootstrap (BS) values of BI analysis.PP ≥ 0.95 and BS values ≥ 85 are plotted on the branches of the tree.PP values are indicated above branches, and BS values are below branches.The specimens of this study are indicated in bold.Branch lengths are scaled to the expected number of substitutions per site.

Figure 1 .
Figure 1.Phylogram based on ITS and LSU sequences of Peltula species; the MrBayes tree is shown with posterior probability (PP) values and bootstrap (BS) values of BI analysis.PP ≥ 0.95 and BS values ≥ 85 are plotted on the branches of the tree.PP values are indicated above branches, and BS values are below branches.The specimens of this study are indicated in bold.Branch lengths are scaled to the expected number of substitutions per site.

Table 1 .
Specimen and sequence information used for molecular phylogenetic analysis.